Soil, Wastewater, Feces and Skin DNA samples for microbiome characterization Raw sequence reads

In this study, we present a novel approach to primer design by truncating both ends of the commonly used 515F and 806R primers for 16S ribosomal RNA gene amplicon sequencing. The design of PCR primers for 16S rRNA gene amplification often involves adding degeneracies to account for mismatches, while keeping the 3-prime end intact to ensure specificity. By truncating primers across the 515F 806R conserved region, we strategically moved the 3-prime end in relation to the standard 19 bp primer site, altering its positional sensitivity to mismatches. By shortening the primers within the conserved region, we posited we could only increase the target range. The standard primers and newly designed primers were tested with a suite of environmental samples, including wastewater, feces, soil and skin DNA extracts. Our results were an equivalency of the truncated primer pool with the standard pool. Additionally, some individual truncated primers showed a greater detection efficiency for Archaea. These results show the viability of using primers much shorter than previously thought possible. This adds more flexibility for future primer design. Our study demonstrates that the truncated primers not only maintain the desired amplification efficiency but also provide broader coverage by avoiding detrimental mismatches, and thereby potentially improving the detection of diverse microbial communities.